Heat stable hyaluronic acid compositions for dermatological use

ABSTRACT

The disclosure provides hyaluronic acid (HA) gel formulations and methods for treating the appearance of the skin. The formulations contain hyaluronic acid and at least one additional ingredient. Methods for treating lines, wrinkles, fibroblast depletions, and scars with the disclosed composition are provided as well.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/099,016 filed on Apr. 14, 2016, which is a continuation of U.S.patent application Ser. No. 13/675,993 filed Nov. 13, 2012, whichgranted as U.S. Pat. No. 9,333,160 on May 10, 2016, which is acontinuation of U.S. patent application Ser. No. 12/714,377 filed Feb.26, 2010, which is abandoned, which is a continuation-in-part of U.S.patent application Ser. No. 12/687,048 filed Jan. 13, 2010, which isabandoned, the entire content of each of which is incorporated herein byreference.

BACKGROUND

Skin aging is a progressive phenomenon, occurs over time and can beaffected by lifestyle factors, such as alcohol consumption, tobacco andsun exposure. Aging of the facial skin can be characterized by atrophy,slackening, and fattening. Atrophy corresponds to a massive reduction ofthe thickness of skin tissue. Slackening of the subcutaneous tissuesleads to an excess of skin and ptosis and leads to the appearance ofdrooping cheeks and eye lids. Fattening refers to an increase in excessweight by swelling of the bottom of the face and neck. These changes aretypically associated with dryness, loss of elasticity, and roughtexture.

A variety of compounds can have an effect on the skin such as wrinklereduction, antioxidant, haemostatic, vasoconstriction, anti-itching,anti-inflammatory and anti-irritant effects. For example, variousvitamins as well and hyaluronic acid (HA) are known to have an effect onskin. Vitamin C is the L-enantiomer of ascorbate and has awell-described role in collagen development. Vitamin C is involved inthe hydroxylation of collagen, which allows it to assume itstriple-helix structure. Vitamin C is also known for its antioxidanteffects and is well tolerated. HA is a natural polysaccharide. It is apolymer of disaccharides that are themselves composed of D-glucuronicacid and N-acetylglucosamine, linked to one another by alternatingbeta-1,4 and beta-1,3 glycosidic linkages. The polymers of thisrecurring unit may be from 10² and 10⁴ kilo Daltons (kDa) in size, invivo. Hyaluronic acid represents a natural constituent of the dermis,where it plays an important role in the hydration and elasticity ofskin. There is a strong correlation between the water content in theskin and levels of HA in the dermal tissue. As skin ages, the amount andquality of HA in the skin is reduced. These changes lead to drying andwrinkling of the skin.

The use of HA in cosmetic and dermatological applications is known. HAis tolerated well and there is no immunogenicity associated with itsuse. The low incidence of side effects has lead to the use of HA for thetreatment of wrinkles, fine lines, and scars. HA is subject todegradation through different pathways (e.g. enzymatic, temperature,free radicals), and therefore, its longevity in vivo is limited.

Disclosures of HA, vitamin C, and C-glycosides include U.S. patentapplication Ser. No. 12/393,884; U.S. Pat. No. 6,921,819 (a process forcross-linking solid hyaluronic acid (HA) by reacting it with apolyfunctional linker during hydration of the HA); U.S. Pat. No.6,685,963 (acrylic particles of HA); U.S. publication 2006/0194758 (amethod for making a hydrogel by cross linking high and low molecularweight sodium HAs); U.S. publication 2009/0036403 (cross-linking HA witha tetra functional PEG epoxide to provide “tunably” cross-linked HA);U.S. publication 2009/0143331 (a HA dermal filler with a degradationinhibitor, such as chondroitin sulphate, in order to provide a longerlasting filler); U.S. publication 2009/0143348 (HA combined with asteroid); and U.S. publication 2009/0155314 (HA combined with abotulinum toxin). Additionally, U.S. publications 2009/0148527,2009/0093755, and 2009/0022808 disclose HA in microspheres, cross-linkedwith collagen, and coated with a protein, respectively. Furtherdisclosures of HA include: WO 2009/034559 (a process for aestheticand/or reparative treatment of the skin with compositions that containat least one C-glycoside derivative); WO 2009/024719 (cosmetic andpharmaceutical compositions that contain HA and a C-glycoside derivativeuseful for filling recesses/depressions in the skin, restore volume ofthe body or the face, and to reduce the sign of aging); WO 2007/128923(a method for preparing a biocompatible gel with controlled release ofone or more active lipophilic and/or amphiphilic ingredients); U.S.publication 2009/0018102 (compositions containing HA and at least oneretinoid or salt/derivative thereof in combination with anoligosaccharide and a HA degradation inhibitor, to treat wrinkles, linesfibroblast depletions and scars); U.S. Pat. No. 3,763,009 (a process forimproving the oxidation resistance of ascorbic acid by subjecting amixture of ascorbic acid, maltose and/or oligosaccharides to an enzymederived from genera Aspergillus, Penicillium or others to enzymaticallyconvert the mixture into ascorbic acid glucoside); U.S. Pat. No.5,616,611 (a α-Glycosyl-L-ascorbic acid that exhibits no direct reducingactivity, is stable, and is useful as a stabilizer, quality-improvingagent, antioxidant, physiologically active agent, a UV-absorbant inpharmaceutical and cosmetic industries); U.S. Pat. No. 5,843,907 (theproduction and use of a crystalline 2-O-α-D-glucopyranosyl-L-ascorbicacid suitable for vitamin C enriching agents, food stuffs,pharmaceuticals, and cosmetics); and EP 0539196 (an industrial scalepreparation of high purity 2-O-α-D-glucopyranosyl-L-ascorbic acid) andUS publication 2002/0151711. Commercial products incorporating HA and/orvitamin C agents include: MESOGLOW® products, REVITACARE®, and NCTF®135/135HA Mesotherapy products. Each of the above-cited references andprinted publications are individually incorporated herein by referencein their entirety.

SUMMARY

Our invention includes a stable dermal filler formulation comprising ahyaluronic acid (HA) and at least one additional ingredient selectedfrom the group consisting of a wrinkle reduction, antioxidant,haemostatic, vasoconstriction, anti-itching, anti-inflammatory andanti-irritant ingredient. Stability of the dermal filler formulation canbe determined by subjecting the dermal filler formulation to a heattreatment selected from the group consisting of (a) steam sterilization(equivalently “autoclaving”) and (b) about 32 days at about 45° C., withsubstantial retention after the heat treatment of one or more of thedermal filler characteristics of being clear, homogenous, and cohesive,and without substantial degradation of the dermal filler formulationafter the heat treatment. Preferably the steam sterilization is carriedout at a temperature of at least about 120° C., as we have found that ahigh temperature steam sterilization reduces the sterilization timerequired while still providing all sterility requirements, withoutdegradation of the dermal filler formulation occurring when theadditional ingredients set forth herein are present in the formulation.More preferably, the steam sterilization is carried out at a temperaturebetween about 130° C. and 135° C., because we found that such aparticular high temperature steam sterilization not only further reducesthe sterilization time required while still providing all sterilityrequirements but as well can be carried out with little or nodegradation of the dermal filler formulation occurring. Preferably, thesteam sterilization is carried out for between about one minute andabout 10 minutes and more preferably for between about 1 minute andabout 5 minutes.

Our invention also includes a steam sterilization stable dermal fillerformulation comprising a hyaluronic acid (HA) and at least oneadditional ingredient selected from the group consisting of wrinklereduction, antioxidant, haemostatic, vasoconstriction, anti-itching,anti-inflammatory and anti-irritant ingredients, wherein the formulationis substantially clear (i.e. little or no modification of the pre-heat[i.e. steam] treatment dermal filler formulation color occurs ascompared to the color of the post heat treatment dermal fillerformulation), homogenous, cohesive stable and not substantially degradedafter steam sterilization. Degradation can be shown after steamsterilization by, for example, discoloration of the steam sterilizeddermal filler formation and/or by a decrease in the homogeneity of theformulation or in other formulation rheological properties.Substantially clear means that on visual inspection the dermal fillerformulation both before and after steam sterilization is not opaque.Substantially homogenous means the dermal filler formulation both beforeand after steam sterilization has the same consistency (eg well mixedthroughout). Substantially monophasic means the dermal fillerformulation both before and after steam sterilization comprises only onephase, meaning it is a gel with no particles. Substantially cohesivemeans the ability of the dermal filler formulation both before and aftersteam sterilization to retain its shape and resist deformation.Cohesiveness is affected by, among other factors, the molecular weightratio of the initial free HA, the degree of crosslinking, the amount ofresidual free HA following crosslinking, and the pH of the dermal fillerformulation. Moreover, a cohesive dermal filler formulation resistsphase separation when tested according to the method disclosed byExample 1A herein.

Our dermal filler formulations are stable after steam sterilization(i.e. at a temperature between about 120° C. to 135° C. or greater).Additionally our dermal filler formulations have long term storage orshelf life stability as shown for example by maintenance of stability ofthe dermal filler formulations in an environment at about 45° C. forabout 32 days (accelerated heat testing), which can be considered toshow that stability will be maintained for about 1 to 3 years at roomtemperature; stability can be determined by substantial retention atroom temperature of one or more of the dermal filler characteristics ofbeing clear, homogenous, and cohesive, and without substantialdegradation of the dermal filler formulation. Stability of our dermalfiller formulations can be determined over a period of or about 25 daysto about 35 days at a temperature of about 35° to 50° C. Preferably, asset forth above, the accelerated heat stability testing is carried outfor about 32 days at about 45° C. Substantially stable after theaccelerated heat (stability) testing carried out as set forth above, orsubstantially stable after autoclaving or after steam sterilization ofthe dermal filler formulation, means the dermal filler formulationretains (as being resistant to degradation) at least 80% and preferablyat least 90% and most preferably at least about 95% of at least one ofits measured characteristics of transparency, pH, extrusion force,rheological characteristics, hyaluronic acid (HA) concentration,sterility, osmolarity, and same additional ingredient concentration. Inour dermal filler formulation the HA is preferably cross-linked and theHA can be present in an amount of about 1 to about 40 mg/mL.

An additional ingredient in our dermal filler formulation can be avitamin B, C or E and the additional ingredient can be present in anamount of about 0.001% to about 10% w/w, and preferably be present in anamount of from about 0.1% to about 3% w/w. Important, the additionalingredient can provide the dermal filler formulation with improvedrheological properties resulting in less extrusion force required foradministration compared to an HA gel formulation without the additionalconstituent.

Our invention also includes a method for treating a dermal conditionsuch as fine lines, wrinkles, fibroblast depletions, and/or scars of apatient by administering to the patient an effective amount of a steamsterilization stable dermal filler formulation comprising a hyaluronicacid (HA) and at least one additional ingredient selected from the groupconsisting of wrinkle reduction, antioxidant, haemostatic,vasoconstriction, anti-itching, anti-inflammatory and anti-irritantingredients, wherein the formulation is clear, homogenous, cohesive,stable and not degraded after steam sterilization and wherein theappearance of the fine lines, wrinkles, fibroblast depletions, or scarsis diminished. The administration can be by sub dermal, intra-dermal orsubcutaneous injected (i.e. local injection administration) into afacial skin of the subject.

Our invention also includes a steam sterilization stable dermal fillerformulation comprising a hyaluronic acid and at least one additionalingredient selected from the group consisting of AA2G and dexpanthenol,wherein the stability of the dermal filler formulation is significantlyincreased by the additional ingredient—as shown by the dermal fillerformulation having a Δ Tan δ 1 Hz<−0.05.

DRAWINGS

FIG. 1 is a representation of the structure of an ascorbyl-2-glucoside,also known as AA2G™ (Hayashibara Co., Japan).

FIG. 2 is a graph showing the synthesis of pro-collagen (% control) forcontrol, gel+lidocaine 0.3%, AA2G™ 0.6% in phosphate buffer, andgel+AA2G™ 0.6%+lidocaine 0.3%.

FIG. 3 is a graph showing the extrusion force over time (3 yr equivalentat 25° C.) in compositions: control, AA2G™ plus lidocaine, and AA2G™plus lidocaine and TPGS.

FIG. 4 is a graph showing the pH over time (3 yr equivalent at 25° C.)in compositions: control, AA2G™ plus lidocaine, and AA2G™ plus lidocaineand TPGS.

FIG. 5 is a graph of tan delta 1 Hz over time (3 yr equivalent at 25°C.) in compositions: control, AA2G™ plus lidocaine, and AA2G™ pluslidocaine and TPGS.

FIG. 6 is an HPLC analysis (C18 column, eluent: sodium phosphate buffer(pH=2.2)/2-propanol 10%, 0.7 ml/min; detection at 260 nm) of AA2G™,lidocaine, and IPA (coeluent) after autoclaving (3 yr equivalent at 25°C.).

FIG. 7 is a graph comparing antioxidant properties in compositions:control versus JUVEDERM® Ultra with lidocaine AA2G™, and JUVEDERM® Ultrawith lidocaine.

DESCRIPTION

Our invention is based on the discovery that a steam sterilizationstable HA based dermal filler can be prepared with an additionalingredient (that is besides the HA present in the formation) which is awrinkle reduction, antioxidant, haemostatic, vasoconstriction,anti-itching, anti-inflammatory and/or anti-irritant ingredient. An HAdermal filler within the scope of our invention (“the dermal fillerformulation”) is (autoclaving) steam sterilization stable and asdemonstrated stability after about 32 days at about 45° C. Theformulation does not exhibit any degradation as shown by the pre andpost autoclaved formulations both being clear, homogenous, and cohesive.

The dermal filler formulation can also exhibit greater stability than anHA gel formulation without the additional constituent. Without wishingto be bound by theory it may be that the matrix of the cross-linked HAused in our formulation sequesters, renders non-reactive and therebyprevents the additional ingredient (as set forth for example in Examples4-6, 10-11, 13, 15-16, 20, 24, and 25-29, supra) from degrading andcauses degradation of the dermal filler formulation during steamsterilization. Additionally, the additional ingredient can behydrophilic and provides protection to the HA from degradation duringsteam sterilization and/or after administration of the dermal fillerformulation to a patient. Without wishing to be bound by theory, theincorporation of an additional ingredient in the dermal fillerformulation may inhibit free-radical scavenging at the injection/implantsite, thereby prolonging dermal filler duration after patientadministration. After steam sterilization the additional ingredient canupon administration (as by subdermal injection) be released from thedermal filler formulation for cosmetic or therapeutic effect.

Autoclave stable or steam sterilization stable as used herein means adermal filler formulation that is resistant to degradation such that theformulation retains at least one, and preferably all, of the followingaspects after steam sterilization: transparent or clear appearance pH,extrusion force and/or rheological characteristics, hyaluronic acid (HA)concentration, osmolarity, and same additional ingredient concentration.

High molecular weight HA as used herein describes a HA material having amolecular weight of at least about 1.0 million Daltons (mw 10⁶ Da or 1MDa) to about 4.0 MDa. For example, the high molecular weight HA in thepresent compositions may have a molecular weight of about 2.0 MDa. Inanother example, the high molecular weight HA may have a molecularweight of about 2.8 MDa.

Low molecular weight HA as used herein describes a HA material having amolecular weight of less than about 1.0 MDa. Low molecular weight HA canhave a molecular weight of between about 200,000 Da (0.2 MDa) to lessthan about 1.0 MDa, for example, between about 300,000 Da (0.3 M Da) toabout 750,000 Da. (0.75 MDa).

Degree of crosslinking as used herein refers to the intermolecularjunctions joining the individual HA polymer molecules, or monomerchains, into a permanent structure, or as disclosed herein the softtissue filler composition. Moreover, degree of crosslinking for purposesof the present disclosure is further defined as the percent weight ratioof the crosslinking agent to HA-monomeric units within the crosslinkedportion of the HA based composition. It is measured by the weight ratioof HA monomers to crosslinker (HA monomers:crosslinker).

Free HA as used herein refers to individual HA polymer molecules thatare not crosslinked to, or very lightly crosslinked to (very low degreeof crosslinking) the highly crosslinked (higher degree of crosslinking)macromolecular structure making up the soft tissue filler composition.Free HA generally remains water soluble. Free HA can alternatively bedefined as the “uncrosslinked,” or lightly crosslinked component of themacromolecular structure making up the soft tissue filler compositiondisclosed herein.

The presence of an additional ingredient in the dermal fillerformulation can provide a stability and longevity that is not exhibitedin a dermal filler formulation containing HA without the additionalingredient. The disclosed formulations after steam sterilization arehomogenous, uncolored, clear, cohesive gel. Our invention includesmethods for treating dermatological conditions, such as fine lines,wrinkles, fibroblast depletions, and/or scars afflicting a subject byadministering to a patient an effective amount of the dermal fillerformulation. The patient can be any mammal, preferably a human of anyage, gender or race. Although typically a subject experiencing the signsof aging skin is an adult, subjects experiencing premature aging orother skin conditions suitable for treatment (for example, a scar) withthe HA gel formulation can be treated as well.

Our dermal filler formulation comprise HA which is preferably at leastpartly cross-linked and can contain some not cross-linked HA. Althoughany pharmaceutically or cosmetically acceptable HA can be used in thedisclosed compositions and formulations, in certain embodiments, thepreferred HA utilized includes those sold as JUVEDERM®, JUVEDERM® 30,JUVEDERM® Ultra Plus, JUVEDERM® Ultra injectable gel (Allergan Inc,Irvine, Calif.). In certain embodiments, the formulation comprises a HAgel matrix and an additional constituent. HA is a known hydrogel. Thegel can be injectable, bioresorbable, monophasic, or biphasic. In someembodiments, the additional constituent can be directly incorporatedinto the HA gel. In other embodiments, in order to increase affinitywith the medium or increase stability, modification of the molecule byderivatization or encapsulation of the constituent can be performed, asdescribed above. For instance, certain oily molecules cannot beintroduced directly into a hydrophilic matrix, and lead to aheterogeneous product. Derivatization of the molecule by graftinghydrophilic moieties is required to increase homogeneity of the gel. Insome embodiments, the gel composition can include a biocompatible orbiodegradable vessel.

The HA gel can be made by any known, suitable methods. Cross-linked HAgels typically have high viscosity and require considerable force toextrude through a fine needle. Uncross-linked HA is often used as alubricant to facilitate the extrusion process. However, especially in HAdermal fillers and implants, uncross-linked HA does not contribute tothe persistence of the final product in vivo. The formulations exhibitincreased stability compared to formulations containing HA without theadditional constituent. Stability is determined by assessing thehomogeneity, color, and clarity, pH, and rheological properties of thegel formulation. The formulations disclosed herein are considered stableif they remain homogenous, colorless, and/or clear, and exhibit stablepH and rheology. The disclosed formulations remain stable for at leastabout 6 months, at least about 1 year, at least about 2 years or atleast about 3 years.

A cross-linking agent can be used to cross-link the HA according to thepresent disclosure. The cross-linking agent may be any agent known to besuitable for cross-linking HA and its derivatives via hydroxyl groups.Suitable cross-linking agents include but are not limited to,1,4-butanediol diglycidyl ether, 1,4-bis(2,3-epoxypropoxy)butane, and/or1,4-bisglycidyloxybutane (commonly known as BDDE),1,2-bis(2,3-epoxypropoxy)ethylene, and1-(2,3-epoxypropyl)-2,3-epoxycyclohexane. The use of more than onecross-linking agent or a different cross-linking agent is included fromthe scope of the present disclosure.

Dermal fillers can be used to treat moderate to severe facial wrinklesand folds such as nasolabial folds (those lines that extend from thenose to the corners of the mouth). In one embodiment, a dermal fillercan be a gel implant formulation that includes HA and an additionalconstituent. The formulations disclosed herein can further includeadditional cosmetic agents that supplement and improve the appearance ofskin. The cosmetic active ingredients may include, but are not limitedto, antioxidants, vitamins, tension agents, and moisturizers.

The formulations disclosed herein can be injected with a syringe intothe mid to deep dermis of the face. The dermis is the subsurface skinlayer that contains connective tissue, nerve endings, and blood vessels.The formulations, when administered as dermal fillers can improve skinappearance by lifting and adding volume to the wrinkles and folds in thetreatment area. Further, in certain embodiments, improvement can be seendue to increased collagen production that results from administration ofthe formulation.

As used herein, “cosmetic” is an adjective referring to improving theappearance of a surface or covering defects. Typically, cosmeticcompositions can be used to improve aesthetic rather than functionalaspects of a surface. Most commonly, cosmetic compositions areformulated for application as a health and beauty treatment or foraffecting personal appearance of the body, for example, keratinoussurfaces such as skin, hair, nails, and the like.

As used herein, “formulation” and “composition” may be usedinterchangeably and refer to a combination of elements that is presentedtogether for a given purpose. Such terms are well known to those ofordinary skill in the art.

Examples of additional ingredients (agents) which can be included in thepresent dermal filler formulations are anti-itch, anti-cellulite,anti-scarring, and anti-inflammatory agents, anesthetics,anti-irritants, vasoconstrictors, vasodilators, as well as agents toprevent/stop bleeding, and improve/remove pigmentation, moisturizers,desquamating agents, tensioning agents, anti-acne agents. Anti-itchagents can include methyl sulphonyl methane, sodium bicarbonate,calamine, allantoin, kaolin, peppermint, tea tree oil, camphor, menthol,hydrocortisone and combinations thereof. Anti-cellulite agents caninclude forskolin, xanthine compounds such as, but not limited to,caffeine, theophylline, theobromine, and aminophylline, and combinationsthereof. Anesthetic agents can include lidocaine, benzocaine, butamben,dibucaine, oxybuprocaine, pramoxine, proparacaine, proxymetacaine,tetracaine, and combinations thereof. Anti-scarring agents can includeIFN-.gamma., fluorouracil, poly(lactic-co-glycolic acid), methylatedpolyethylene glycol, polylactic acid, polyethylene glycol andcombinations thereof. Anti-inflammatory agents can includedexamethasone, prednisolone, corticosterone, budesonide, estrogen,sulfasalazine, mesalamine, cetirizine, diphenhydramine, antipyrine,methyl salicylate, loratadine, and derivatives and combinations thereof.Additionally, active agents such as epinephrine, thymidine, cytidine,uridine, antiypyrin, aminocaproic acid, tranexamic acid, eucalyptol,allantoin, glycerin, and sodium selenite, can be included. The discloseddermal filler formulations can further comprise degradation inhibitors.Degradation inhibitors, include but are not limited to,glycosaminoglycans (e.g., heparin, heparin sulfate, dermatan sulfate,chondroitin sulfate, o-sulfated HA, linamarin, glucosamine, andamygdalin), antioxidants (e.g. ascorbic acid, melatonin, vitamin C,vitamin E, sodium selenite, glutathion, retinoic acid, coenzyme,beta-carotene, allopurinol, mannitol, caffeic acid, caffeine,polyphenol, theobromine, catechin), proteins (e.g., serum hyaluronidaseinhibitor), and fatty acids (e.g. saturated C₁₀ to C₂₂ fatty acids),vitamin B and complex, and combinations thereof as noted, in certainembodiments, the additional ingredient can be an antioxidant. In certainembodiments, the antioxidant comprises a vitamin C such asascorbyl-2-glucoside (available as AA2G™, Hayashibara Co., Japan) (FIG.1), and/or a vitamin E such as d-alpha-tocopheryl polyethylene glycol1000 succinate (TPGS). Anti-irritants can include thymol, bisabolol.Healing agents can include allantoin, eucalyptol, chitosane, cytidine,thimidine, uridine, lanoline. Anti-bleeding: epinephrine,norepinephrine, phenylephrine, synephrine, naphazoline, aminocaproicacid, tranexamic acid, ethamsylate, vitamin K. Collagen promoters caninclude retinol, peptide sequences. Additionally, active ingredients(agents) such as epinephrine, thymidine, cytidine, uridine, antipyrine,aminocaproic acid, eucalyptol, sodium selenite, can be included.

In some embodiments, the HA is present at a concentration of about 1 toabout 40 mg/mL, or about 10 to about 40 mg/mL, or about 20 to about 30mg/mL. In certain embodiments, the HA is present in a concentration ofabout 20 to about 25 mg/mL. In certain embodiments, the HA is present ata concentration of 24 mg/mL. The additional constituent can be presentin an amount of about 0.001 to about 10% w/w, or from about 0.001 toabout 5% w/w, or from 0.3 to about 3% w/w.

In certain embodiments, the disclosure provides a dermal fillercomprising (a) about 90 wt %, or about 95 wt %, or about 100 wt % of ahigh molecular weight (about 1 million to about 3 million Daltons) HA;and (b) 0 wt %, or about 5 wt %, or about 10 wt % of a low molecularweight (less than 1 million Daltons) HA. In certain embodiments, the HAis present in the dermal filler at a concentration of about 10 to about24 mg HA/mL dermal filler and the HA is about 4% to about 11%cross-linked. In certain embodiments, the cross linker is 4-butane dioldiglycidyl ether (BDDE). The dermal filler can further comprise about0.1 wt % or 0.6 wt %, or 1.0 wt % of an ascorbyl-2-glucoside, such asAA2G™ (Hayashibara, Japan). In a preferred embodiment, 0.6 wt % AA2G™(i.e., 6 mg AA2G™/g HA) is utilized and renders a concentration of2.1012 mM AA2G™.

Topical formulations of AA2G™ are known. However, there are nosubdermally administered formulations of AA2G™ available, which islikely due to the fact that a topical AA2G™ is not thought to lenditself to an injectable formulation. The disclosure provides the firstinjectable formulation of AA2G™ that is efficacious, compatible, andstable over time.

The disclosed compositions are also well suited for mesotherapy.Mesotherapy is a non-surgical cosmetic treatment technique involvingintra-epidermal, intra-dermal, and/or subcutaneous injection of an agent(micronutrients, vitamins, mineral salts, etc). The compositions areadministered in the form of small multiple droplets into the epidermis,dermo-epidermal junction, and/or the dermis.

The formulations of the disclosure can be injected utilizing needleswith a diameter of about 0.26 to about 0.4 mm and a length ranging fromabout 4 to about 14 mm. Alternately, the needles can be 21 to 32 G andhave a length of about 4 mm to about 70 mm. Preferably, the needle is asingle-use needle. The needle can be combined with a syringe, catheter,and/or a pistol (for example, a hydropneumatic-compression pistol).

The formulations can be administered once or over several sessions withthe subject spaced apart by a few days, or weeks. For instance, thesubject can be administered a formulation every 1, 2, 3, 4, 5, 6, 7,days or every 1, 2, 3, or 4, weeks. The administration can be on amonthly or bi-monthly basis. Further, the formulation can beadministered every 3, 6, 9, or 12 months.

Our dermal filler formulation can optionally include one or more agentssuch as, without limitation, emulsifying agents, wetting agents,sweetening or flavoring agents, tonicity adjusters, preservatives,buffers antioxidants and flavonoids. Tonicity adjustors useful in apharmaceutical composition of the present disclosure include, but arenot limited to, salts such as sodium acetate, sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjusters. Preservatives useful in the dermal fillerformulation described herein include, without limitation, benzalkoniumchloride, chlorobutanol, thimerosal, phenyl mercuric acetate, and phenylmercuric nitrate. Various buffers and means for adjusting pH can be usedto prepare the dermal filler formulation, including but not limited to,acetate buffers, citrate buffers, phosphate buffers and borate buffers.Similarly, antioxidants useful in the dermal filler formulation includefor example, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Flavonoids arecompounds found in plants that are well known to have diverse beneficialbiochemical and antioxidant effects. Subcategories of flavonoidsinclude: flavones, flavonols, flavanones and flavanonols. Examples offlavonoids include: luteolin, apigenin, tangeritin, quercetin,kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin,hesperetin, naringenin, eriodictyol, homoeriodictyol, taxifolin,dihydroquercetin, dihydrokaempferol, tannic acid, tannins, condensedtannins, and hydrolysable tannins. It is understood that these and othersubstances known in the art can be included in the dermal fillerformulations disclosed herein. The pH of the disclosed dermal fillerformulations can be about 5.0 to about 8.0, or about 6.5 to about 7.5.In certain embodiments, the pH of the formulation is about 7.0 to about7.4 or about 7.1 to about 7.3.

A dermal filler formulation must be capable of withstandingsterilization which is a strict requirement before the product can besold (the product must be sterile). Sterilization can be carried out bysteam sterilization, filtration, microfiltration, gamma radiation, ETOlight or by a combination of these methods. It is known that a dermalfiller can be steam sterilized (autoclaved) without degradation ofphysical properties, but when a dermal filler formulation contains anadditional labile ingredient (such as an anti-oxidant, wrinklereduction, haemostatic, vasoconstriction, anti-itching,anti-inflammatory, and/or anti-irritant ingredient, such as a vitamin,vitamin derivative or analgesic compound) the entire dermal fillerformulation or at least the additional (heat labile) ingredient issterilized by a non-heat treatment such as by a filtration sterilizationmethod. Thus, the known dermal filler product (“Revitacare”) is sold intwo separate vials or containers, one vial containing the HA (which isautoclave sterilized)) and the second vial containing any additionalingredients (the second vial contents are sterilized by filtration).Another known dermal filler product NCTF®135 HA is sold in a singlecontainer holding both HA and any additional ingredients, all havingbeen sterilized by microfiltration. It is an important aspect of ourinvention that we mix the HA and the additional ingredients and thenautoclave the completed dermal filler formulation with maintenance ofgel properties (i.e. non-degraded and storage stable formulation).Additionally we have discovered dermal filler formulations that exhibitretention of stability after being treated (accelerated heat testenvironment) to about 45° C. for about 30 days, or at least about 60days, or at least about 90 days with no degradation of physicalproperties.

To reiterate an important aspect of our invention and a significantdistinction over known dermal fillers is that our dermal fillerformulations are prepared by: (1) mixing the HA and the additionalingredient(s), and then; (2) autoclaving (no filtration sterilization ofany component) the complete dermal filler formulation with; (3)maintenance of the desired gel properties (no degradation of any dermalfiller constituent or ingredient, and stable).

EXAMPLES

In the Examples below autoclaving means steam sterilization carried outat a temperature between about 130° C. to about 135° C. for betweenabout one minute and about 10 minutes.

Example 1A Method for Determining Gel Cohesivity

For purposes of example only and not to be considered as limiting thepresent invention in any way, the following tests may be performed inorder to evidence or quantify cohesivity of a HA-based gel composition.

First, 0.2 g or 0.4 g of a gel composition to be tested is placed in aglass syringe. Next, 0.2 g or more of phosphate buffer is added to thesyringe and the mixture is thoroughly mixed for about 1 hour to obtain ahomogenous mixture. Then, the homogenized mixture is centrifuged for 5min at 2000 tr/min to remove the air bubbles and to allow thedecantation of any particles. The syringe is then held in a verticalposition and one drop of eosin colorant is deposited at the surface ofthe gel by means of a syringe and an 18G needle. After 10 min, the dyehas slowly diffused through the gel.

After dilution of the gel, homogenization and decantation, a relativelylow cohesivity gel shows a phase separation (an upper diluted lessviscous phase without particles and a lower one composed of decantedparticles that are visible with the naked eye or under microscope).Under the same conditions, a highly cohesive gel shows substantially nophase separation, and the dye is prevented from diffusing into thecohesive formulation. A relatively less cohesive gel, on the other hand,shows a clear phase separation.

Example 1 Properties of Formulations of NaHA and Water Soluble Moleculesare Tested

The active ingredient was incorporated into a NaHA matrix andautoclaved. The properties of the gel, aspect (i.e.,color/clarity/homogeneity) and extrusion force were analyzed aftersterilization at 3 years equivalent at room temperature. Table 1 showsthat all formulations were clear, homogenous, and uncolored at the3-year mark. The extrusion forces after autoclaving and at 3 yearsequivalent at room temperature are shown as well. In conclusion, theincorporation of the molecules has no impact on gel properties andingredient structure.

TABLE 1 Extrusion force (N) Content Extrusion force (N) 3 years~roomIngredient (%) Aspect after autoclaving T° C. Allantoin 0.3 Clear PASSEDPASSED 0.5 Homogeneous PASSED PASSED Cytidine 0.5 Uncolored PASSEDPASSED 1 PASSED PASSED Thymidine 0.5 PASSED PASSED 1 PASSED PASSEDUridine 0.5 PASSED PASSED 1 PASSED PASSED Antipyrin 0.5 PASSED PASSED 1PASSED PASSED Aminocaproic acid 0.5 PASSED PASSED 1 PASSED PASSEDTranexamic acid 0.5 PASSED PASSED Eucalyptol 0.5 PASSED PASSED Sodiumselenite 0.1 PASSED PASSED Glycerin 0.5 PASSED PASSED

Acceptance criteria: “Passed” means that the change of extrusion force(ΔF) was less than two Newtons (<2 N). In other words the measured ΔF ofthe extrusion force of the HA gel with the specified ingredients minusthe extrusion force of the HA gel without the added ingredients was <2N.

Example 2 Preparation of NaHA Gel Containing Vitamin C

Ascorbic acid (1% w/w) was incorporated into a NaHA matrix. (JUVEDERM®FORMA). The pH was adjusted to about 7 and composition was autoclaved.The gel obtained was clear, yellow and degraded.

Example 3 Alternative Preparation of NAHA Gel Containing Vitamin C

Magnesium Ascorbyl Phosphate (MAP) (0.6%, 1 or 2% w/w) was incorporatedin a NaHA matrix (JUVEDERM® Ultra). The pH was adjusted to about 7 andthe compositions were autoclaved. All gels obtained were uncolored andclear. The gel properties after autoclaving are shown in Table 2.Extrusion force acceptance criteria: Conform with NaHA matrixspecifications.

TABLE 2 After autoclaving Formulation Extrusion force (N) JUVEDERM ®Ultra + 0.6% MAP PASSED JUVEDERM ® Ultra + 1% MAP PASSED JUVEDERM ®Ultra + 2% MAP PASSED

Rheology data of the gel containing 2% MAP after autoclaving is shown inTable 3. Rheological properties are followed as a function of time usinga controlled stress rheometer according to the following method:frequency sweep from 0.05 to 10 Hz with 0.8% controlled strain. Adegradation of the gel was observed by rheology. TAN δ×HZ is arheological characterisation which shows the ratio of viscous modulus toelastic modulus. It shows the degradation of the gel.

Δ Tan δ1 Hz=(Tan δ1 Hz formulation)−(Tan δ1 Hz NaHA matrix)

Acceptance criterion: Δ Tan δ 1 Hz<0.1

TABLE 3 Formulation Δ Tan δ 1 Hz JUVEDERM ® Ultra + 2% MAP 0.344

Example 4 Alternative Preparation of NAHA Gel Containing Vitamin C

Sodium Ascorbyl Phosphate (SAP) (0.6%, 1% and 2% w/w) was incorporatedin an NaHA matrix (JUVEDERM® Ultra). The pH was adjusted to about 7 andthe composition was autoclaved. All gels obtained were uncolored andclear. The gel properties after autoclaving are shown in Table 4.

TABLE 4 After autoclaving Formulation Extrusion force (N) JUVEDERM ®Ultra + 0.6% SAP PASSED JUVEDERM ® Ultra + 1% SAP PASSED JUVEDERM ®Ultra + 2% SAP PASSED

Rheology data of the gel containing 2% SAP after autoclaving is shown inTable 5. No degradation of the gel was observed by rheology.

TABLE 5 Formulation Δ Tan δ 1 Hz JUVEDERM ® Ultra + 2% SAP 0.089

Example 5 Alternative Preparation of NaHA Gel Containing Vitamin C

Ascorbic acid 2-Glucoside (AA2G™) at a concentration of 0.6%, 1% and 2%w/w was incorporated in an NaHA matrix (JUVEDERM® Ultra Plus). The pHwas adjusted to about 7 and the composition was autoclaved. All gelsobtained were uncolored and clear. The gel properties after autoclavingare shown in Table 6.

TABLE 6 After autoclaving Formulation Extrusion force (N) JUVEDERM ®Ultra Plus + 0.6% AA-2G PASSED JUVEDERM ® Ultra Plus + 1% AA-2G PASSEDJUVEDERM ® Ultra Plus + 2% AA-2G PASSED

The gels containing 0.6%, 1% and 2% were stable (pH, injection force)after autoclaving. Rheology data of the gels containing 0.6%, 1% and 2%w/w AA2G™ after autoclaving is shown in Table 7. No degradation of thegel was observed by rheology at each AA2G™ concentration.

TABLE 7 Formulation Δ Tan δ 1 Hz JUVEDERM ® Ultra Plus + 0.6% AA2G ™−0.010 JUVEDERM ® Ultra Plus + 1% AA2G ™ −0.014 JUVEDERM ® Ultra Plus +2% AA2G ™ −0.016

Rheological studies showed an slightly increase of the stability of thegel in the presence of the additive.

Example 6 Effect of Vitamin C on Aspect and Stability of the Gel

The shelf-life at 45° C. during 32 days was tested for the formulationsprepared in example 5 and the NaHA matrix JUVEDERM® Ultra Plus. Rheologydata of the gels containing 0.6%, 1% and 2% of AA2G™ are shown in Table8.

TABLE 8 Formulation Δ Tan δ 1 Hz JUVEDERM ® Ultra Plus + 0.6% AA2G ™−0.050 JUVEDERM ® Ultra Plus + 1% AA2G ™ −0.045 JUVEDERM ® Ultra Plus +2% AA2G ™ −0.059

The gels containing ascorbyl glucoside maintained their properties afterautoclaving and over a period of 32 days at 45° C. SurprisingRheological studies showed an increase of the stability of the gel inthe presence of the additive.

Example 7 Preparation of NaHA Gel Containing Vitamin E

Tocopheryl Acetate (0.5% w/w) was incorporated into a NaHA matrix.(JUVEDERM® 30) and autoclaved. The gel obtained was unclear, white.

Example 8 Alternative Preparation of NaHA Gel Containing Vitamin E

Sodium Tocopheryl Phosphate (STP), at 0.4%, 1.2% w/w, was incorporatedin a NaHA matrix (JUVEDERM® FORMA) and autoclaved. The gel obtained wasnot clear (white).

Example 9 Alternative Preparation of NaHA Gel Containing Vitamin E

Polyoxyethanyl-α-tocopheryl sebacate (0.7% w/w) was incorporated in aNaHA matrix (JUVEDERM® Ultra Plus) and autoclaved. The gel obtained wasclear, but heterogenous.

Example 10 Alternative Preparation of NaHA Gel Containing Vitamin E

Tocopherol polyethylene glycol 1000 succinate (TPGS) was incorporated invarying concentrations (1%, 3.5% and 7% w/w) in a NAHA matrix (JUVEDERM®FORMA) and autoclaved. “JUVEDERM® FORMA” means the Juvederm formulationwas used. All gels obtained were uncolored and clear. The gel propertiesafter autoclaving are shown in Table 9.

TABLE 9 Formulation Extrusion force (N) JUVEDERM ® FORMA + 1% TPGSPASSED JUVEDERM ® FORMA + 3.5% TPGS PASSED JUVEDERM ® FORMA + 7% TPGSPASSED

Rheology data of the gels containing 1%, 3.5% and 7% TPGS afterautoclaving is shown in Table 10. No degradation of the gel was observedby rheology at each TPGS concentration.

TABLE 10 Formulation Δ Tan δ 1 Hz JUVEDERM ® FORMA + 1% TPGS 0.008JUVEDERM ® FORMA + 3.5% TPGS −0.007 JUVEDERM ® FORMA + 7% TPGS −0.011

These rheological studies showed the stability of the dermal fillerformulation with a particular additional ingredient.

Example 11 Stability of Formulations Containing Additional Ingredients

The stability of various formulations was tested. The ingredients shownin Table 11 were incorporated into a NaHA matrix, and autoclaved. Thedegradation of the formulations after autoclaving is shows in Table 11and after 48 days at 45° C. in Table 12. The stability of extrusionforce, pH, and degradation are shown over time in FIGS. 3, 4, and 5,respectively. HPLC analysis (C18 column; eluent: sodium phosphate buffer(pH 2.2), 2-propanol 10%, 0.7 ml/min; detection at 260 nm) confirmed theingredients after autoclaving and 3-year shelf-life are shown in FIG. 6.

TABLE 11 Δ Tan δ 1 Hz After autoclaving 45° C., 48 days JUVEDERM ® UltraPlus + 0.059 0.020 AA2G ™ 0.6% + Lidocaine 0.3% JUVEDERM ® Ultra Plus +0.016 0.007 AA2G ™ 0.6% + TPGS 1.5% + lidocaine 0.3%

Example 12 AA2G™ Promotes Collagen Synthesis

Human skin fibroblasts were cultured in a 12 wells plate. At confluence,100 μL of each compound (Juvederm® FORMA with 0.3% lidocaine, Juvederm®FORMA+AA2G™ 0.6%+Lidocaine 0.3% and Phosphate Buffer with 0.6% AA2G) wasdeposited in a culture insert (porosity of 0.4 μm), which was itselflaid on the fibroblast monolayers. In parallel, a control withouttreatment was performed. Cultures were incubated for 72 hours and eachexperimental condition was conducted done in triplicate. At the end ofincubation, cell viability was verified by microscopic observation andMTT reduction assay. Pro-collagen I secretion was measured using ELISAkit. The presence of 0.6% AA2G™ in a hyaluronic acid gel containing 0.3%lidocaine increased pro-collagen synthesis by a factor 3 (+292%),whereas JUVEDERM® gel with 0.3% lidocaïne showed an increase of 40% ofthe pro-collagen secretion (see FIG. 2).

Example 13 AA2G™ Protects NaHA from Oxidative Degradation

The effect of AA2G™ on NaHA oxidative degradation was studied. Oxidationtesting was used as it allows testing of the resistance of a NaHA matrixto free radicals. Degradation by free radicals was simulated on arheometer (Haake Rheostress 600) by addition of 1/7 ratio of H₂O₂ 30% onthe surface of a spread gel measured with a controlled stress rheometeraccording to the following method: frequency of 1 Hz with 0.8%controlled strain, during 3600 s at 35° C. The time value is taken at 5Pa/s.

Further, a comparison of antioxidant properties for JUVEDERM® Ultra withAA2G™ 0.6%/Lidocaine 0.3% formulation (15 800 s) versus NaHA matrixJUVEDERM® Ultra with Lidocaine (4 942 s) showed that the gel containingAA2G™ and lidocaine is more stable with respect to free radical activity(see FIG. 7). AA2G™ protected against oxidative degradation by a factorof 3.

Example 14 Implantation Study

A gel containing AA2G™ at 0.6% (=6 mg/g=2.10⁻² mM) was implanted in thedeep dermis and subcutaneous tissues in rats. Histological evaluation at1 week showed some mononuclear cells (lymphocytes and plasmocytes)around the implants in all implantation sites (test and control). Theywere also associated with macrophages. The gel containing AA2G™ appearedto be less inflammatory. The irritation index in test samples(AA2G™+NaHA) was 9.9 compared to 12.3 in controls (NaHA only). Table 12shows the histological results at 1 week, 1 month, and 3 months. Theirritation score of AA2G™ gel are (for each implantation time) lowerthan control (gel without AA2G™)

TABLE 12 NAHA + AA2G + Lido Biocompatibility ISO 10993 Cytotoxicity ✓(non cytotoxic) Irritation ✓ (non irritant) Sensitization ✓ (nonsensitizing) Implantation Test 1 week ✓ (no skin reaction) 3 weeks ✓ (noskin reaction) 3 months ✓ (no skin reaction)

Example 15 Incorporation of Dexpanthenol in NaHA Gel Formulations

Dexpanthenol was incorporated into a NaHA matrix JUVEDERM® Ultra Pluswith Lidocaine (with 0.3% w/w lidocaine) with a content of 1% w/w. Thegel was autoclaved. The gel obtained was clear and uncolored before andafter autoclaving. The gel properties after autoclaving are shown inTable 13.

TABLE 13 After autoclaving Extrusion force Formulation (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus with PASSED 0.026 Lidocaine (0.3%) + Dexpanthenol1%

Example 16 Effect of the Incorporation of Dexpanthenol in NAHA GelFormulations

The shelf-life at 45° C. during 30 days was tested of the formulationsprepared in example 15 and the NaHA matrix JUVEDERM® Ultra Plus XC. Thegel was clear, uncolored. Rheology data of the gels containingdexpanthenol 1% w/w and lidocaine 0.3% w/w are shown in Table 14.

TABLE 14 After 30 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus with −0.071 Lidocaine (0.3%) + Dexpanthenol 1%

Rheological studies showed an increase of the stability of the gel inthe presence of the additive.

Example 17 Incorporation of Epinephrine in NaHA Gel Formulations

Epinephrine was incorporated into a NaHA matrix (JUVEDERM® Ultra Plus)with a 10 ppm epinephrine bitartrate. The gel was autoclaved. The gelobtained was clear and uncolored before and after autoclaving. The gel(dermal filler formulation) properties after autoclaving are shown inTable 15.

TABLE 15 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus + PASSED 0.165 epinephrine bitartrate 10 ppm

Degradation of the gel was observed by rheological analysis.

Example 18 Incorporation of Epinephrine in NaHA Gel Formulations

Epinephrine was incorporated into a NaHA matrix (JUVEDERM® Ultra Plus)with 0.3% lidocaine and 10 ppm epinephrine bitartrate. The gel wasautoclaved. The gel obtained was clear and colored after autoclaving.The gel properties after autoclaving are shown in Table 16.

TABLE 16 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus + PASSED 0.092 Lidocaine 0.3% + epinephrinebitartrate 10 ppm

A slight degradation of the gel was observed by rheological analysis.

Example 19 Effect of Additional Ingredient on the Stability of GelContaining Epinephrine and Lidocaine

The shelf-life at 45° C. during 60 days was tested of the formulationsprepared in Example 18 and the NaHA matrix JUVEDERM® Ultra Plus. The gelwas clear, slightly colored. Rheology data of the gels containingepinephrine bitartrate (10 ppm), lidocaine (0.3% w/w) is shown in Table17.

TABLE 17 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus + 0.185 Lidocaine 0.3% + epinephrine bitartrate 10 ppm

After a stability of 60 days at 45° C., the gel containing epinephrineand lidocaine was unstable.

Example 20 Incorporation of Epinephrine in NaHA Gel FormulationsContaining an Antioxidant

Epinephrine was incorporated into a NaHA matrix (JUVEDERM® Ultra Plus)with epinephrine bitartrate (10 ppm) and mannitol (0.9 or 4.5% w/w). Thegels were autoclaved. The gel with 4.5% mannitol was clear and uncoloredbefore and after autoclaving whereas with 0.9% mannitol was slightlycolored. The gel properties after autoclaving is shown in Table 18.

TABLE 18 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus + PASSED 0.047 epinephrine bitartrate 10 ppm +mannitol 0.9% JUVEDERM ® Ultra Plus + PASSED 0.015 epinephrinebitartrate 10 ppm + mannitol 4.5%

No degradation was observed for either of the dermal filler formulationstested.

Example 21 Effect of Additional Ingredient on the Stability of GelContaining Epinephrine and an Antioxidant

The shelf-life at 45° C. during 60 days was tested of the formulationsprepared in example 20 and the NaHA matrix JUVEDERM® Ultra Plus. Thegels were clear, slightly colored. Rheology data of the gels containingepinephrine bitartrate (10 ppm) and mannitol (0.9 or 4.5% w/w) is shownin Table 19.

TABLE 19 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus + 0.061 epinephrine bitartrate 10 ppm + mannitol 0.9%JUVEDERM ® Ultra Plus + 0.006 epinephrine bitartrate 10 ppm + mannitol4.5%

After a stability of days at 45° C., both gels containing epinephrine,lidocaine and mannitol were stable. The composition containing 4.5%mannitol was more stable.

Example 22 Incorporation of Epinephrine in NaHA Gel FormulationsContaining Lidocaine and Antioxidant

Epinephrine was incorporated into a NaHA matrix (JUVEDERM® Forma) withepinephrine bitartrate (20 ppm), lidocaine (0.3% w/w) and mannitol (4.5%w/w). The gel was autoclaved. The gel obtained was clear slightlycolored after autoclaving. The gel properties after autoclaving areshown in Table 20.

TABLE 20 After autoclaving Formulation Extrusion force (N) Δ Tan δ 1 HzJUVEDERM ® Forma + PASSED 0.026 Lidocaine 0.3% + epinephrine bitartrate20 ppm + mannitol 4.5%

No degradation was observed.

Example 23 Effect of Additional Ingredient on the Stability of GelContaining Epinephrine, Lidocaine and an Antioxidant

The shelf-life at 45° C. during 60 days was tested of the formulationsprepared in example 22 and the NaHA matrix JUVEDERM® Forma. The gel wasclear, slightly colored. Rheology data of the gel containing epinephrinebitartrate (20 ppm), lidocaine (0.3% w/w) and mannitol (4.5% w/w) isshown in Table 21.

TABLE 21 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Forma + −0.030 epinephrine bitartrate 20 ppm + mannitol 4.5%

The gel (dermal filler formulation) was stable after 60 days at 45° C.

Example 24 Incorporation of Synephrine in NaHA Gel FormulationsContaining Lidocaine and Antioxidant

Synephrine was incorporated into a NaHA matrix Juvederm Ultra Plus withLidocaine (with 0.3% w/w lidocaine) with a content of 100 ppm ofsynephrine. The gel was autoclaved. The gel obtained was clear anduncolored before and after autoclaving. The gel properties afterautoclaving is shown in Table 22.

TABLE 22 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® with lidocaine (0.3%) + PASSED −0.006 synephrine 100 ppm

Example 25 Effect of Additional Ingredient on the Stability of GelContaining Synephrine and Lidocaine

The shelf-life at 45° C. during 60 days was tested of the formulationsprepared in example 24 and the NaHA matrix JUVEDERM® Ultra Plus withLidocaine. The gels was clear, uncolored. Rheology data of the gelcontaining synephrine 100 ppm and lidocaine 0.3% w/w is shown in Table23.

TABLE 23 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus with lidocaine −0.028 (0.3%) + synephrine 100 ppm

This rheological study showed maintenance of the stability of the gel(dermal filler formulation) in the presence of the particular additionalingredient (additive) shown.

Example 26 Incorporation of Phenylephrine in NaHA Gel FormulationsContaining Lidocaine

Phenylephrine was incorporated into a matrix JUVEDERM® Ultra Plus withLidocaine (with 0.3% w/w lidocaine) with a content of 100 ppmphenylephrine. The gel was autoclaved. The gel obtained was clear anduncolored before and after autoclaving. The gel properties afterautoclaving are shown in Table 24.

TABLE 24 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus with PASSED −0.002 Lidocaine 0.3% + Phenylephrine100 ppm

Example 27 Effect of Additional Ingredient on the Stability of GelContaining Phenylephrine and Lidocaine

The shelf-life at 45° C. during 60 days was tested of the formulationsprepared in example 26 and the NaHA matrix JUVEDERM® Ultra Plus withLidocaine. The gel was clear, uncolored. Rheology data of the gelcontaining phenylephrine 100 ppm and lidocaine 0.3% w/w are shown inTable 25.

TABLE 25 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus with −0.017 Lidocaine (0.3%) + Phenylephrine 100 ppm

This rheological study showed maintenance of the stability of the gel(dermal filler formulation) in the presence of the particular additionalingredient (additive) shown.

Example 28 Incorporation of Naphazoline in NaHA Gel FormulationsContaining Lidocaine and Antioxidant

Naphazoline was incorporated into a matrix Juvederm Ultra Plus withLidocaine (with 0.3% w/w lidocaine) with a content of 100 ppm. The gelwas autoclaved. The gel obtained was clear and uncolored before andafter autoclaving. The gel properties after autoclaving are shown inTable 26.

TABLE 26 After autoclaving Extrusion Formulation force (N) Δ Tan δ 1 HzJUVEDERM ® Ultra Plus with PASSED −0.003 Lidocaine (0.3%) + Naphazoline100 ppm

Example 29 Effect of Additional Ingredient on the Stability of GelContaining Naphazoline and Lidocaine

The shelf-life at 45° C. over 60 days was tested of the formulationsprepared in example 28 and the NaHA matrix JUVEDERM® Ultra Plus withLidocaine. The gel was clear, uncolored. Rheology data of the gelcontaining naphazoline 100 ppm and lidocaine 0.3% w/w is shown in Table27.

TABLE 27 After 60 days at 45° C. Formulation Δ Tan δ 1 Hz JUVEDERM ®Ultra Plus with −0.008 Lidocaine 0.3% + Naphazoline 100 ppm

Example 30 Treatment Example

A woman, age 37, presents with fine lines around her eyes and deeperwrinkles on the sides of her mouth. She receives injections of aformulation of Example 10. She receives the injections in the fine linesand in the wrinkles once a week for 3 weeks and notices a visibleimprovement in the appearance of her skin.

Example 31 Alternate Treatment Example

A 59 year old man presents with wrinkles between his eyebrows and in thenasolabial folds. He receives injections of the dermal fillerformulation of Example 11, every 3 months. A visible improvement in thewrinkles is seen.

Example 32 Alternate Treatment Example

A 35 year old woman presents with fine lines across her forehead. Shereceives injections of the dermal filler formulation of Example 15, oncea week for two weeks, and notices an improvement in the appearance ofthe skin on her forehead.

Example 33 Alternate Treatment Example

A woman, age 44, presents with uneven texture on her right cheekresulting from a loss of collagen due to aging. She receives injectionsof the dermal formulation of Example 20 (using the 4.5% mannitol dermalfiller formulation), in her cheek to build up the areas where thecollagen has been lost. A visible improvement is seen in the texture ofthe skin on her cheek after 3 series of injections over a 2 week periodof time.

Example 34 Alternate Treatment Example

A 35 year old man presents with a deep wrinkle across his chin and finelines on the sides of his eyes. He receives the dermal fillerformulation of Example 26 along the sides of his eyes. He receives 2series of injections in his chin, spaced 1 week apart. The fine linesand wrinkle are visibly diminished after treatment.

Example 35 Alternate Treatment Example

A woman, age 62, presents with wrinkles across her forehead, on thesides of her eyes, in the nasolabial folds, and a scar on her chin. Shereceives injections of the dermal filler formulation of Example 29 eachweek for one month. After the injections, the appearance of the wrinklesand the scar is visibly diminished.

Our results above show that at least each of the two additionalingredients AA2G and dexpanthenol significantly increased the stabilityof the dermal filler formulation (HA gel), as shown by the dermal fillerformulation having a Δ Tan δ 1 Hz<−0.05.

With regard to dexpanthenol: panthenol is the alcohol analog ofpantothenic acid (vitamin B5), and is thus the provitamin of B5 which invivo is oxidized to pantothenate. Panthenol is a highly viscoustransparent liquid at room temperature, but salts of pantothenic acid(for example sodium pantothenate) are powders (typically white).Panthenol is soluble in water, alcohol and propylene glycol, soluble inether and chloroform, and slightly soluble in glycerin. Panthenol hastwo D and L enantiomers with only the D enantiomer (D-panthenol, alsocalled dexpanthenol) being biologically active, however both the D and Lforms have moisturizing properties. For topical cosmetic use panthenolhas been used in the D form and as a racemic mixture of D and L(DL-panthenol). Thus topical dexpanthenol cream (sold under the genericname “panthoderm”) is made by mixing with an emollient and has been usedfor relieving dry skin, preventing and treating sore nipples duringbreast-feeding, and promoting healing of burns and poorly-healingwounds.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein. Furthermore,numerous references have been made to patents and printed publicationsthroughout this specification. Each of the above-cited references andprinted publications are individually incorporated herein by referencein their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described. Specificembodiments disclosed herein may be further limited in the claims usingconsisting of or consisting essentially of language. When used in theclaims, whether as filed or added per amendment, the transition term“consisting of” excludes any element, step, or ingredient not specifiedin the claims. The transition term “consisting essentially of” limitsthe scope of a claim to the specified materials or steps and those thatdo not materially affect the basic and novel characteristic(s).Embodiments of the invention so claimed are inherently or expresslydescribed and enabled herein.

We claim:
 1. A steam sterilization-stable dermal filler formulationcomprising a crosslinked hyaluronic acid (HA), ascorbyl-2-glucoside andan anesthetic agent.
 2. The dermal filler formulation of claim 1,wherein the hyaluronic acid is crosslinked with 1,4-butanedioldiglycidyl ether.
 3. The dermal filler formulation of claim 1, whereinthe anesthetic agent is lidocaine.
 4. The dermal filler formulation ofclaim 3, wherein the lidocaine is present in an amount of about 0.001%to about 10% w/w.
 5. The dermal filler formulation of claim 3, whereinthe lidocaine is present in an amount of about 0.001% to about 5% w/w.6. The dermal filler formulation of claim 3, wherein the lidocaine ispresent in an amount of about 0.3% to about 3% w/w.
 7. The dermal fillerformulation of claim 3, wherein the lidocaine is present in an amount of0.3% w/w.
 8. The dermal filler formulation of claim 1, wherein thecrosslinked HA is present in an amount of about 1 mg/mL to about 40mg/mL.
 9. The dermal filler formulation of claim 8, wherein thecrosslinked HA is present in an amount of about 10 mg/mL to about 40mg/mL.
 10. The dermal filler formulation of claim 8, wherein thecrosslinked HA is present in an amount of about 20 mg/mL to about 30mg/mL.
 11. The dermal filler formulation of claim 8, wherein thecrosslinked HA is present in an amount of about 20 mg/mL to about 25mg/mL.
 12. The dermal filler formulation of claim 1, wherein theascorbyl-2-glucoside is present in an amount of about 0.001% w/w toabout 10% w/w.
 13. The dermal filler formulation of claim 12, whereinthe ascorbyl-2-glucoside is present in an amount of about 0.1% w/w toabout 3.0% w/w.
 14. The dermal filler formulation of claim 12, whereinthe ascorbyl-2-glucoside is present in an amount of 1% w/w.
 15. Thedermal filler formulation of claim 12, wherein the ascorbyl-2-glucosideis present in an amount of 2% w/w.
 16. The dermal filler formulation ofclaim 12, wherein the ascorbyl-2-glucoside is present in an amount of0.6% w/w.
 17. The dermal filler formulation of claim 1, wherein thesteam-sterilization stability of the dermal filler formulation isdetermined by subjecting the dermal filler formulation to a steamsterilization treatment at between about 120° C. and about 135° C. forbetween about 1 minute and about 5 minutes, with substantial retentionafter the treatment of one or more of the dermal filler characteristicsof being clear, homogenous and cohesive.
 18. The dermal fillerformulation of claim 17, wherein the HA is crosslinked with1,4-butanediol diglycidyl ether, and the anesthetic agent is lidocaine.19. The dermal filler formulation of claim 18, wherein the crosslinkedHA is present in an amount of about 10 mg/mL to about 40 mg/m L.
 20. Thedermal filler formulation of claim 19, wherein the ascorbyl-2-glucosideis present in an amount of about 0.1% w/w to about 3.0% w/w, and thelidocaine is present in an amount of about 0.3% to about 3% w/w.